Directly injectable formulations which provide enhanced cryoprotection of cell products

ABSTRACT

This invention provides compositions and methods for cryoprotection of recombinant live cancer cells. Specifically, an improved cryoprotective medium is provided which includes a hydroxyethyl starch and/or derivative thereof alone or in combination with either DMSO or glycerol.

FIELD OF THE INVENTION

[0001] The invention relates to compositions and methods for thecryopreservation of cellular vaccines. More specifically, the inventionrelates to the use of particular compositions comprising cryoprotectivemedia that includes hydroxyethyl starch (HES) and glycerol or DMSOtogether with recombinant live cancer cells. The compositions can befrozen and thawed, when desired, for use as a cellular vaccine for thetreatment of cancer.

BACKGROUND OF THE INVENTION

[0002] At certain periods of time in a tumor's cell growth, thepatient's immune system has the ability to recognize the growth asabnormal (or non-self). As a result, various methods have been developedwhich take advantage of the patient's own immune system to fight cancer.Exemplary methods include the use of polyclonal and monoclonalantibodies, non-specific immune system stimulants, such as cytokines,protein or peptide subunit vaccines (e.g., using antigens that are oftenassociated with cancer cells, such as tumor-specific antigens andtumor-associated antigens), adoptive immunotherapy or cellular therapy,gene therapy, cellular vaccines, etc. See, e.g., WO 00/3387.

[0003] Cellular vaccines wherein cells (or derivatives thereof are thetherapeutic agent are currently in clinical testing for treatment ofcancer. Such cellular vaccines provide advantages over isolated proteinvaccines in that whole cells are the vehicle for a broad range ofantigens, e.g., on the cell surface. See, for example, Dranoff et al. WO93/06867, Gansbacher et al., WO 94/18995, Jaffee et al WO 97/24132,Mitchell et al. WO 90/03183 and Morton et al WO91/06866, each of whichis expressly incorporated by reference herein.

[0004] Cells for use in such cellular vaccines can be modified, e.g. toexpress a protein which modulates the immune response to the cell. Forexample, a gene encoding a cytokine or costimulatory molecule may beintroduced into cells derived from, e.g., a primary tumor taken from apatient, to create recombinant cancer cells. When the cytokine orcostimulatory molecule is expressed, it is capable of modulating theimmune response to the cell. The recombinant cancer cells may beexpanded in vitro, treated to prevent further growth and returned to thepatient. Appropriate timing of administration(s) and good cell viabilityare required for effective treatment with a cellular vaccine. Thus, itis important to be able to store recombinant cancer cells for use atselected time points, appropriate to particular treatments. Storage andmaintenance of viability are important in order to allow fortransportation, to decrease the amount of cell divisions the cellsundergo before use in treatment and to ensure that an adequate andreproducible dose is delivered to the patient.

[0005] Freezing of cell compositions with maintenance of viability hasbeen the subject of considerable research. Maintenance of cell viabilityfollowing freezing and thawing continues to be a challenge. In thefreezing process as the liquid component of a cell is changed to asolid, ice crystals are formed and damage can occur to the cells. Atleast two types of damage to cells is possible when ice crystals areformed. Rapid growth of ice crystals may physically disrupt membranesand subcellular organelles and may even lyse cells. Slow growth of icecrystals may result in cellular dehydration (because of the exclusion ofelectrolytes from the ice crystals) and extra-cellular ice formation.See, e.g., Gorlin, (1996) Journal of Infusional Chemotherapy,6(1):23-27.

[0006] In an attempt to minimize the effects of ice crystal formation,cells are typically frozen in medium with cryoprotectants.Cryoprotectants protect the cells during freezing in a variety of ways.Collagiative cryoprotectants penetrate the cell and decrease the osmoticgradient across membranes. Vitrifying cryoprotectants increase the glassformation of the solution thereby creating a glass wall around the cell,which prevents dehydration. Cryoprotectants can also work by inhibitingice crystal formation. See, e.g., Gorlin, (1996) Journal of InfusionalChemotherapy, 6(1):23-27.

[0007] Different cell types vary in their permeability to water and intheir sensitivity to solute concentration. Leibo et al., (1970)Cryobiology, 6(4): 315-332. As a result, different types andcombinations of cryoprotectants have been found to be effective topreserve specific types of cells. For example, human bone marrowcommitted stem cells have been shown to be preserve by a cryoprotectantcombination of dextran, glycerol, and dimethyl sulfoxide (Odavic et al.(1980) Experienta 36:1122-1124), and mouse marrow stem cells have beenshown to be preserved by polyvinylpyrolidone (PVP), sucrose or glycerol.See, e.g., Stiff et al. (1987) Blood, 70(4): 974-978; Venkataraman,(1997) Cryobiology, 34:276-283; Wang et al., (1998) Cryobiology,37:22-29; Merten et al., (1995) Biologicals, 23:185-189; and, Yoshidaand Takeuchi, (1991) Cytotechnology 5:99-106. Polymers, such ashydroxyethyl starch (HES), have been used to cryoprotect human monocytesand unfractionated cells for use in bone marrow transplantation. See,e.g., Takashi et al., (1988) Biophysical Journal, 54:509-518; and Stiffet al. (1987) Blood, 70(4): 974-978.

[0008] Cryoprotective media for cellular vaccines has not been reported.Thus, there remains a need for cryoprotective media and procedures thatcan be used to successfully preserve cellular vaccines for use astherapeutic agents. In view of the above, materials and methods thatwould provide for successful preservation and recovery of cells with ahigh percentage viability following freezing and thawing would be highlydesirable for use in cellular vaccines. The present invention addressesthis need, as will be apparent from the detailed description providedherein.

SUMMARY OF THE INVENTION

[0009] The invention provides compositions and methods that includecryoprotective media and recombinant live cancer cells for protectingand preserving the cells during freezing and thawing. The cryoprotectivemedium includes a hydroxyethyl starch (HES) or derivative thereof aloneor combination with DMSO or glycerol.

[0010] Compositions of the invention include a recombinant live cancercell in a cryoprotective medium comprising about 5% by weight to about22% by weight of a hydroxyethyl starch (HES) or derivative thereof aloneor in combination with about 1% by weight to about 15% by weight of DMSOor glycerol, and, optionally, from about 0% by weight to about 10% byweight human serum albumin (HSA).

[0011] A composition can include about 8% by weight of HES or aderivative thereof and about 5% by weight of DMSO or glycerol, and,optionally, about 2% by weight human serum albumin (HSA).

[0012] The HES of the invention can include a variety of molecules. Forexample, HES molecules can be: (a) etherified with hydroxyethyl groupswherein the degree of molecular substitution is from about 0.60-0.80;(b) etherified with hydroxyethyl groups, wherein the degree of molecularsubstitution is from about 0.40-0.60; or a combination of (a) and (b).Other examples of HES, include, but are not limited to HES moleculesthat are: (a) etherified with hydroxyethyl groups wherein the degree ofmolecular substitution is about 0.70; (b) etherified with hydroxyethylgroups, wherein the degree of molecular substitution is from about 0.4to about 0.5; or a combination of (a) and (b). In one embodiment, HESincludes a molecule with (a) a molecular weight of from about 420 toabout 480 kDa; (b) a molecular weight of from about 200 to about 290kDa; or a combination of (a) and (b).

[0013] Many types of recombinant live cancer cells, such aspatient-derived tumor cells, autologous cells, allogeneic cells andbystander cells, respectively, can be used in practicing the invention.Typically, a recombinant live cancer cell for use in practicing theinvention comprises an introduced heterologous nucleic acid codingsequence or gene for a cytokine or costimulatory molecule.

[0014] Methods for preserving viability of recombinant live cancer cellsare also included within the invention. Such methods include obtaining aplurality of recombinant live cancer cells, concentrating the cells,suspending the cells in a cryoprotective medium such as described above,thereby providing a suspension and freezing the suspension to acryogenic frozen state, thereby providing a frozen suspension. Afterthawing of the suspension to a liquid state at least about 60%, about65%, about 70%, about 75% or more of the recombinant live cancer cellsremain viable. In one embodiment, the recombinant live cancer cells areirradiated prior to freezing or in the cryogenic frozen state, whereinthe suspension is frozen at a temperature from about −200° C. to atemperature of about −35° C. The methods further include thawing thefrozen suspension and administering the thawed suspension to a subject.Optionally, the thawed suspension is not washed prior to theadministering step.

[0015] The invention further includes a frozen or thawed suspensionprepared by a method described herein.

[0016] Kits for preserving cell viability are also included in theinvention. Kits of the invention include: a container containing acryoprotective medium such as described above and instructionalmaterials teaching the use of the cryoprotective medium to preserve cellviability.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a graph showing the % viability of LNCaP cells before(pre-freeze), immediately after (initial thaw) and 24 hours after thecells were suspended in cryoprotective media, frozen to −80° C. andthawed at 37° C. The cryoprotective media tested was as follows: 1×PBSalone; 5% glycerol alone; 24% HSA, 5% glycerol; 5% HSA, 5% glycerol;saturated HES in 5% glycerol/5%. HSA/PBS;20% Hetastarch in 5%glycerol/5% HSA/PBS;20% Pentastarch in 5% glycerol/5% HSA/PBS; 15% PEG(10K) in 5% glycerol/5% HSA/PBS; Dextran 50K in 5% glycerol/5% HSA/PBS.

[0018]FIGS. 2A and 2B are graphs showing the viable cell recovery (%viability) of LNCaP cells either immediately following freezing at −80°C. and thawing at 37° C. (initial thaw) or 24 h after thawing (24 hrlater). The LNCaP cells were suspended in cryoprotective mediacomprising polymer 1B (20% HES) alone with or without a second polymer(FIG. 2A) or polymer 1B (20% HES) plus 5% glycerol with or without asecond polymer (FIG. 2B).

[0019]FIGS. 3A and 3B are graphs showing the % viability (FIG. 3A) andGM-CSF secretion (FIG. 3B), of several independently produced culturesof PC-3 cells, each of which was suspended in 20% HES +5% DMSO or 5%glycerol, frozen to −80° C., then thawed at 37° C.

[0020]FIGS. 4A and 4B are graphs showing the % viability (FIG. 4A) andGM-CSF secretion (FIG. 4B) of several independently produced cultures ofLNCaP cells, each of which was suspended in 20% HES +5% DMSO orglycerol, frozen at −80° C., then thawed at 37° C. FIG. 5 is a graphshowing the % viable cell recovery of LNCaP cells immediately followingfreezing at −80° C. and thawing at 37° C. or 24 h after thawing. Thecells were frozen in cryoprotective medium comprising from 8% to 26% HESand 5% glycerol in PBS. FIG. 6A is a graph showing the % viability ofPC-3 cells in a variety of cryoprotective media at time-zero (the timeof freezing) and after 3 months, 6 months and 12 months storage at −80°C. The formulations tested are indicated in the figure.

[0021]FIG. 6B is a graph showing the GM-CSF secretion by PC-3 cells attime-zero (the time of freezing) and after 3 months, 6 months and 12months storage at −80° C. in a variety of cryoprotective media. Theformulations tested are indicated in the figure.

DETAILED DESCRIPTION

[0022] There are many benefits to freezing cells in multiple aliquotsand preserving their viability when thawed. For example, the frozencells can be stored and/or transported for future use, e.g., fortreatment, such as a cellular vaccine. However, freezing cells andpreserving their viability is not a simple matter. Cell types differ intheir permeability to water and in their sensitivity to soluteconcentration, thus, different types and combinations of cryoprotectantsare needed to preserve specific types of cells. The invention providescomposition and methods for preserving the viability of recombinantcancer cells using a cryoprotective medium that includes a hydroxyethylstarch (HES) or a derivative thereof alone or in combination with DMSOor glycerol.

Definitions

[0023] Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. It is to be understood that this invention is not limitedto the particular methodology, protocols, and reagents described, asthese may vary. For the purpose of the present invention, the followingterms are defined below.

[0024] The term “autologous” refers to cancer cells derived from anindividual or primary descendents of those cells, wherein the cells maybe used as a cellular vaccine for that same individual.

[0025] The term “allogeneic” as used herein refers to cancer cells ofthe same type being harbored by an individual, but established from acancer cell line derived from an unrelated individual. Alternatively, anallogeneic cancer cell line is derived from an unrelated tumor type tothe tumor harbored by an individual, but which shares common tumorassociated antigens with the tumor of that individual. In general,allogeneic refers to genetic differences within a species, that is,differences between individuals of the same species. It follows thatallogeneic cells for use as a cellular vaccine are geneticallydissimilar to those of the individual to which they are administered.

[0026] The term “bystander” as used herein refers to a mammalian cellline, preferably a human cell line, which naturally lacks majorhistocompatibility class I (MHC-I) antigens and major histocompatibilityclass II (MHC-II) antigens or is modified so that it lacks MHC-Iantigens and MHC-II antigens. Theoretically, any mammalian, preferablyhuman, cell line that is capable of paracrine production of a cytokineor costimulatory molecule can be used. An exemplary bystander cell lineis K562 (ATCC CCL-243; Lozzio et al., (1975) Blood 45(3): 321-334; Kleinet al. (1976) Int. J. Cancer 18: 421-431). Heterologous nucleic acids orgenes that encode a cytokine or costimulatory molecule may be introducedinto bystander cells, and they may be mixed with cancer cells for use ina cellular vaccine.

[0027] By “introduced” is meant the provision to an autologous,allogeneic or bystander cell line of a nucleic acid molecule, e.g., avector, that comprises a heterologous nucleic acid coding sequence orgene for a cytokine or costimulatory molecule that either is notexpressed in the cell line or, as a result of the provision of thenucleic acid molecule, is now expressed at a greater level. A “vector”encompasses a DNA molecule, such as a plasmid, virus or other vehicle,which contains one or more heterologous or recombinant DNA sequences,e.g., a cytokine or costimulatory molecule gene or coding sequence ofinterest, under the operative control of a functional promoter and insome cases an enhancer as well. By recombinant or heterologous withreference to a vector or other DNA sequences merely acknowledges thelinkage of DNA sequences which are not typically conjoined as found innature.

[0028] The term “patient-derived tumor cells” refers to cells that havebeen recovered from a tumor taken from a patient. Typically a cellsuspension is created from the isolated tumor. When used as a cellularvaccine, patient-derived tumor cells are treated in a manner effectiveto stop further replication, i.e. the cells are irradiated.

[0029] The term “recombinant cancer cell” refers to a cancer cell inwhich a heterologous nucleic acid coding sequence or gene for a cytokineor costimulatory molecule have been introduced.

[0030] The term “vaccine”, as used herein refers to a cellularcomposition (e.g., a recombinant live cancer cell) for administration toa patient as part of a therapeutic regimen for the treatment of cancer.The vaccine typically contains cancer cells, some or all of which havebeen genetically modified to express a cytokine or other costimulatorymolecule.

[0031] The term “cryogenic frozen state” refers to a temperature atwhich the cells can be frozen and/or stored for a desired length oftime. For example a cryogenic frozen state can refer to a temperature,e.g., from about −200° C. to a temperature of about −35° C., from about−180° C. to a temperature of about −35° C., from about −150° C. to atemperature of about −35° C., from about −200° C. to a temperature ofabout −50° C., from about −200° C. to a temperature of about −60° C. andthe like.

[0032] The term “cryoprotective medium” refers to the medium in whichcells are suspended when frozen and/or thawed. For example, thecryoprotective medium described herein includes a hydroxyethyl starch(HES) or derivative thereof alone or in combination with DMSO orglycerol. The cryoprotective medium can also include human serum albumin(HSA). The term “cryoprotective medium” may be used interchangeably withthe terms “cryo-protecting formulation” and “cryoprotectiveformulation”. The components of the “cryoprotective medium” may bedescribed herein as “polymers”.

[0033] The terms “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein refer to at least two nucleotides or analoguesthereof, covalently linked together. A nucleic acid of the invention istypically single-stranded or double stranded and will generally containphosphodiester bonds, although in some cases, nucleic acid analogs areincluded that can have alternate backbones, comprising, for example,phosphoramide; phosphorothioate, O-methylphophoroamidite linkages orpeptide nucleic acid backbones and linkages. The depiction of a singlestrand also defines the sequence of the other strand and thus alsoincludes the complement of the sequence.

[0034] The term “hydroxyethyl starch” or “HES”, as used herein refers toa polymer of hydroxyethyl starch or a derivative thereof, which exhibitsthe same properties as a cryoprotectant as HES itself. HES of theinvention can include a variety of molecules, as further describedbelow.

[0035] The term “patient” as used herein may refer to any mammal. Theinvention is useful for both the human and other mammalian subjects.

[0036] Cryoprotective Medium

[0037] The invention provides cryoprotective formulations for preservingthe viability of recombinant live cancer cells during freezing to acryogenic frozen state and thawing to a liquid state. These preservedrecombinant live cancer cells can be used, when desired, as cellularvaccines, e.g., for the treatment of cancer.

[0038] Cryoprotective medium of the invention includes at least onecryoprotectant, such as, hydroxyethyl starch (HES), and often includesother cryoprotectants as well, e.g., DMSO, or glycerol, and optionallyhuman serum albumin (HSA). In one embodiment, the cryoprotective mediumof the invention can be free or substantially free of human serumalbumin (HSA). Since HSA is a human-derived material, elimination ofthis excipient can reduce the potential risk of contamination ofcellular product with adventitious agents.

[0039] The cryoprotective medium of the invention includes a polymer,hydroxyethyl starch (HES). HES has typically found use as a plasmaexpander. Hetastarch, the most common hydroxyethyl starch, is derivedfrom corn starch and has a molecular weight of about 450,000 daltons.Pentastarch is an analog of Hetastarch with a molecular weight of about264,000 daltons. The HES of the invention includes a variety ofmolecules. For example, HES can comprise HES molecules that are (a)etherified with hydroxyethyl groups where the degree of molecularsubstitution is from about 0.60-0.80; (b) etherified with hydroxyethylgroups, wherein the degree of molecular substitution is from about0.40-0.60; or a combination of (a) and (b). Other examples of HES,include, but are not limited to, HES that is, e.g., (a) etherified withhydroxyethyl groups where the degree of molecular substitution is about0.70; (b) etherified with hydroxyethyl groups, where the degree ofmolecular substitution is from about 0.4 to about 0.5; or a combinationof (a) and (b). In one embodiment, HES includes a molecule with (a) amolecular weight of from about 420 to about 480 kDa; (b) a molecularweight of from about 200 to about 290 kDa; or a combination of (a) and(b). For example, HES can include Hetastarch (B. Braun-McGaw) and/orPentastarch (B. Braun-McGaw).

[0040] HES has been used as a protective agent for the cryopreservationof erythrocytes and platelets and when combined with DMSO. See, e.g.,Ashwood-Smith et al. (1972) Cryobiology 9:441-449.

[0041] Dimethylsulfoxide (DMSO) or glycerol are also optionally used inthe cryoprotective media of the invention. While the mechanism is notpart of the invention, DMSO and glycerol can act by decreasing theosmotic gradient across membranes. These agents maintain an increasedvolume of cellular solution and exert colligative (depending on thenumber of particles (and not on the nature of the particles) actionwithin the cells. This avoids an excessive concentration of toxicelectrolytes in the non-frozen cellular solution. If there is enoughprotective agent compound, the salt concentration does not rise to acritically damaging level until the temperature becomes so low that thedamaging reactions are slow enough to be tolerated by the cells.

[0042] In one aspect, a composition of the invention includes arecombinant live cancer cell in a cryoprotective medium, wherein thecryoprotective medium comprises a hydroxyethyl starch (HES) orderivative thereof alone or in combination with DMSO. In one embodiment,the composition includes about 5% by weight to about 22% by weight ofHES or a derivative thereof and about 1% by weight to about 15% byweight of DMSO, and, optionally, from about 0% by weight to about 10% byweight human serum albumin (HSA). In another embodiment, a compositioncan include about 5% by weight to about 15% by weight of HES or aderivative thereof and about 1% by weight to about 10% by weight ofDMSO, and, optionally, from about 0% by weight to about 10% by weighthuman serum albumin (HSA). In another embodiment, a composition caninclude about 5% by weight to about 10% by weight of HES or a derivativethereof and about 1% by weight to about 6% by weight of DMSO, and,optionally, from about 0% by weight to about 10% by weight human serumalbumin (HSA). For example, in one preferred embodiment, a compositionof the invention includes a recombinant live cancer cell in acryoprotective medium comprising a final percentage of about 8% byweight HES, about 2% by weight HSA, and about 5% by weight DMSO. HES mayinclude Pentastarch, Hetastarch, a combination or derivative thereof.

[0043] Compositions of the invention also include a recombinant livecancer cell in a cryoprotective medium, wherein the cryoprotectivemedium comprises a hydroxyethyl starch (HES) or derivative thereof aloneor in combination with glycerol. In one embodiment, a compositionincludes about 5% by weight to about 22% by weight of the HES orderivative thereof and about 1% by weight to about 15% by weight of theglycerol, and, optionally, from about 0 to about 10% human serum albumin(HSA). In another embodiment, a composition includes about 5% by weightto about 15% by weight of the HES or derivative thereof and about 1% byweight to about 10% by weight of the glycerol, and, optionally, fromabout 0 to about 10% human serum albumin (HSA). In yet anotherembodiment, a composition includes about 5% by weight to about 10% byweight of the HES or derivative thereof and about 1% by weight to about6% by weight of the glycerol, and, optionally, from about 0 to about 10%human serum albumin (HSA). For example, in another preferred embodiment,a composition of the invention includes a recombinant live cancer cellin a cryoprotective medium comprising a final percentage of about 8% byweight HES, about 2% by weight HSA, and about 5% by weight glycerol. HESmay include Pentastarch, Hetastarch, a combination or derivativethereof.

[0044] A cryoprotective medium of the invention is typically diluted ina solution, which is at a physiological pH. Exemplary solutions for usein practicing the invention include, but are not limited to phosphatebuffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), IDMEM,MEM, RPMI 1640, Ham's F-12, Normosol R, lactated Ringer's, Hank'sbalanced salt solution (HBSS), and combinations thereof. In addition,the cryoprotecive medium can contain auxiliary substances, such as,water, saline, pH buffering agents, carriers or excipients, otherstabilizers and/or buffers or other reagents that enhance the viabilityand/or cytokine or other costimulatory molecule expression of therecombinant live cancer cells following the freezing and thawingprocess.

[0045] Typically, the cell suspension is frozen at a temperature of frome.g., from about −200° C. to a temperature of about −35° C., from about−180° C. to a temperature of about −35° C., from about −150° C. to atemperature of about −3520 C., from about −200° C. to a temperature ofabout −50° C., from about −200° C. to a temperature of about −60° C. andthe like. In one embodiment, frozen cells are stored at a temperaturebelow which any recrystallization can occur, e.g., the temperature belowthe glass transition of pure water (e.g., less than about −135° C.).Standard methods known in the art are used to freeze the cells, e.g.,containers holding a cryoprotective medium of the invention andrecombinant cells can be immersed in, a solid carbon dioxide and alcoholmixture, or in liquid nitrogen. The containers can also be placeddirectly in a freezer, which is set at a desired temperature, e.g.,about equal to or less than about −35° C. Cryogenic equipment can alsobe used, e.g., a programmed freezer or rate-controlled freezer(Cryo-Med, Mt. Clemens, Mich. or UTL-80, Neslab Instruments Inc.,Portsmouth, N.H.). The frozen cells are typically transferred to afreezer, which is set at a desired temperature, e.g., about equal to orless than about −35° C. Liquid nitrogen (the liquid and/or gas phase)can also be used to freeze and store the cells. Other freezing methodsand apparatus known in the art can also be used in practicing themethods described herein.

[0046] Methods for preserving viability of recombinant live cancer cellsupon freezing and thawing are included in the invention. An exemplarymethod includes the steps of obtaining a plurality of recombinant livecancer cells (e.g., mammalian cells, such as lung cancer cells,pancreatic cancer cells, prostate cancer cells, kidney cancer cells,myeloma cells, leukemic cells and the like); concentrating the pluralityof recombinant live cancer cells using standard procedures routinelyemployed in the art; suspending the recombinant live cancer cells in acryoprotective medium comprising a HES or a derivative thereof alone orin combination with DMSO and from about 0% by weight to about 10% byweight human serum albumin (HSA), thereby providing a suspension. Thesuspension is frozen to a cryogenic frozen state, thereby providing afrozen suspension, where following thawing of the suspension to a liquidstate at least about 60%, 65%, 70%, 75% or more of the recombinant livecancer cells remain viable. In one approach, the recombinant live cancercells are irradiated (e.g., irradiated prior to freezing or irradiatedin the cryogenic frozen state). Typically, the frozen suspension isfrozen at a temperature of from about −200° C. to a temperature of about−35° C.

[0047] The recombinant cancer cells are typically expanded in vitro,treated to render the cells proliferation-incompetent and returned tothe patient. In one embodiment, the cells are renderedproliferation-incompetent by irradiation. Typically, the cells areplated in a tissue culture plate and irradiated at room temperatureusing a Cs source and irradiated at a dose rate of from about 50 toabout 200 rads/min, preferably, from about 120 to about 140 rads/min. Ina preferred approach, the cells are irradiated with a total dosesufficient to inhibit the majority of cells from proliferating in vitro.Thus, desirably the cells are irradiated with a total dose of from about10,000 to 20,000 rads, optimally, with about 15,000 rads, such that 100%of the cells are rendered proliferation-incompetent.

[0048] In one embodiment, a frozen suspension prepared a methoddescribed herein is included in the invention. In another embodiment, athawed suspension prepared by a method described herein is included inthe invention.

[0049] In one embodiment, the methods further include thawing the frozensuspension thereby providing a thawed suspension and administering thethawed suspension to a subject. Optionally, the thawed suspension is notwashed prior to the administering step.

[0050] Thawing can take place by allowing samples to thaw slowly at,e.g., room temperature, or by immersing frozen samples in liquid, e.g.,water, at a set temperature, e.g., about 37° C. Cells can also be thawedby mixing the cells with thawed medium. Optionally, cells can be thawedusing a programmed freezer.

[0051] In one embodiment, the plurality of recombinant live cancer cellsare autologous or allogeneic and further comprise an introducedheterologous nucleic acid coding sequence for a cytokine orcostimulatory molecule, e.g., granulocyte-macrophage colony stimulatingfactor (GM-CSF). In another embodiment, the recombinant live cancercells are bystander cells, and the method further comprises thawing thefrozen suspension, thereby providing a thawed suspension; providingpatient-derived tumor cells, mixing the thawed suspension withpatient-derived tumor cells (e.g., irradiated tumor cells), therebyproviding a mixed suspension and administering the mixed suspension to asubject.

[0052] Methods of the invention obtaining a plurality of recombinantlive cancer cells (e.g., autologous cells, allogeneic cells, bystandercells and the like); concentrating the plurality of recombinant livecancer cells; suspending the plurality of recombinant live cancer cellsin a cryoprotective medium, comprising about 8% by weight of HES or aderivative thereof, about 2% by weight HSA and about 5% by weight DMSO,thereby providing a suspension; freezing the suspension to a cryogenicfrozen state, thereby providing a frozen suspension; and irradiating theplurality of live recombinant cancer cells or the frozen suspension,where following thawing of the suspension to a liquid state at leastabout 60%, about 65%, about 70%, about 75% or more of the recombinantlive cancer cells remain viable. In one embodiment, the method furtherincludes thawing the frozen suspension, thereby providing a thawedsuspension and administering the thawed suspension to a subject wherethe thawed suspension is not washed prior to the administering step. Athawed suspension prepared by this method is also included in theinvention.

[0053] Following freezing and thawing, the thawed recombinant livecancer cells are returned, delivered, or transferred to the subject. Thecells may be delivered to the tissue site from which they were obtainedor to another site appropriate to the therapeutic regimen. If desired,the cells can be grafted onto a tissue, skin, organ, or body system ofinterest in the subject using standard and well-known graftingtechniques or delivered to the blood or lymphatic system using standarddelivery or transfusion techniques. Such delivery, administration, ortransfer of thawed recombinant live cancer cells are typically made byusing one or more of the routes or modes of administration describedherein and know in the art. In one aspect, the amount of thawedrecombinant live cancer cells administered is sufficient and effectiveto treat the disease or condition at the site or tissue system. Thethawed recombinant live cancer cells can be administered, for example,intramuscularly, intradermally, subdermally, subcutaneously, orally,intraperitoneally, intrathecally, intravenously, or placed within acavity of the body (including, e.g., during surgery), or by inhalationor vaginal or rectal administration.

[0054] Typically, the thawed recombinant live cancer cells (s) areadministered in an amount sufficient to induce or reduce a desiredphenotype, i.e., an “effective amount”. Single or multipleadministrations of the thawed recombinant live cancer cells can becarried out as needed. The subject can be at any stage of development atthe time of administration, e.g., embryonic, fetal, infantile, juvenileor adult.

[0055] Recombinant Cancer Cells

[0056] The recombinant live cancer cells used in practicing theinvention find utility in active immunotherapy which involves theinjection of cancer or tumor cells in vivo to generate either a novel oran enhanced immune response thereto. The tumor cells employed can bepatient-derived tumor cells (autologous or allogeneic), or bystandercells. Many types of recombinant live cancer cells find use inpracticing the invention. Typically, the recombinant live cancer cellsinclude an introduced heterologous nucleic acid coding sequence or genefor a cytokine or costimulatory molecule. Exemplary cytokines orcostimulatory molecules include, but are not limited to, any moleculewhich is involved with the initiation, enhancement, strengthening,heightening, and/or lengthening of an immune response. In someembodiments, the cytokine or costimulatory molecule is GM-CSF, IL-1,IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, CD2, IL-12, IL-15, IL-18, TGF, B7,MIP-1a, MIP-1p, MIP-2, M-CSF, G-CSF, and/or ICAM. In one preferredembodiment, the cytokine is GM-CSF. In other embodiments, more than onecytokine or costimulatory molecule is expressed by a recombinant livecancer cell of the invention.

[0057] In one embodiment, a heterologous nucleic acid coding sequence orgene is introduced into a cancer cell, e.g., a lung cancer cell, apancreatic cancer cell, a prostate cancer cell, a kidney cancer cell, amyeloma cell, a leukemic cell and the like. Various methods can beemployed for delivering a nucleic acid molecule, e.g., a vector, to acell in vitro or ex vivo. Live cancer cells of the invention aretypically genetically engineered (e.g., transformed, transduced ortransfected) with a heterologous nucleic acid coding sequence. Manyapproaches for introducing nucleic acids into cells are known in theart. Such methods include electroporation, membrane fusion withliposomes (lipofection), high velocity bombardment with DNA, calciumphosphate mediated transfection, DEAE-dextran mediated transfection,infection with viral vectors, by use of polycation compounds such aspolylysine, receptor specific ligands direct microinjection into singlecells, spheroplast fusion whereby E coli containing the nucleic acidmolecules are stripped of their outer cell walls and fused to animalcells using polyethylene glycol and the like. See, Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (“Berger”), Sambrook et al.,Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”), andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds., JohnWiley & Sons, Inc., (supplemented through 2002), each of which isexpressly incorporated by reference herein.

[0058] In vectors for use in practicing the invention, the heterologousnucleic acid coding sequence or gene for a cytokine or costimulatorymolecule is operably linked to a promoter that is capable of drivingexpression of the coding sequence or gene. A coding sequence or gene is“operably linked” when the promoter is capable of directingtranscription of the coding sequence or gene. A “gene” is any nucleicacid sequence coding for a protein or mRNA molecule. A gene comprisescoding sequences and non-coding (e.g., regulatory) sequences, while a“coding sequence” is limited to coding DNA. A “promoter” is a DNAsequence that directs the binding of RNA polymerase and thereby promotesRNA synthesis. “Enhancers” are cis-acting elements of DNA that stimulateor inhibit transcription of adjacent genes. All proper transcription,translation and processing signals (e.g., splicing and polyadenylationsignals) are correctly arranged on the vector such that the cytokine orcostimulatory molecule gene or coding sequence is appropriatelytranscribed and translated in the cell into which it is introduced. Theconsrution of such vectors for effective expression in host cells iswell within the knowledge of the ordinary skilled artisan.

[0059] As/used herein, a cytokine or costimulatory molecule gene orcoding sequence includes genomic or cDNA sequences, variant sequencesand mutations thereof, whether isolated from nature or synthesized inwhole or in part, so long as the gene or coding sequence can express aprotein having the characteristic function of the cytokine,costimulatory molecule. The means of modifying genes or coding sequencesare well-known in the art.

[0060] Any vector can be employed that is suitable for introduction ofnucleic acids into eukaryotic cells, or more particularly animal cells,such as mammalian, e.g., human, cells. Preferably, the vector iscompatible with the cell, e.g., is capable of effecting expression ofthe cytokine or costimulatory gene or coding sequence in the cell.Exemplary vectors for use in practicing the invention include, but arenot limited to, viruses, plasmids, retrotransposons, cosmids and/or thelike. Viral vectors include, but are not limited to parvovirus vectors,herpes virus vectors, retrovirus vectors, adenovirus vectors, lentiviralvectors, and the like. Alone, or in combination with viral vectors, anumber of non-viral vectors are also useful for introducing (e.g.,transfecting) heterologous nucleic acid coding sequences for a cytokineor costimulatory molecule into cells. Suitable non-viral vectorsinclude, but are not limited to, plasmids, cosmids, and phagemids,liposomes, water-oil emulsions, polethyleneimines, biolisticpellets/beads, and dendrimers.

[0061] In one aspect of the invention, ex vivo methods are employedwherein a plurality of cells are taken from an individual, aheterologous nucleic acid coding sequence or gene for a cytokine orcostimulatory molecule is introduced into the cells in a mannereffective to express the cytokine or costimulatory molecule, as furtherdescribed above.

[0062] Ex vivo methods are typically employed with cells that areautologous or allogeneic. A heterologous nucleic acid coding sequence orgene for a cytokine or costimulatory molecule may be introduced intosuch autologous or allogeneic cells, followed by expression thereof,resulting in production of a recombinant live cancer cell. Theautologous or allogeneic recombinant live cancer cell can be frozenusing the cryoprotective medium and methods described herein. Whenthawed, the recombinant live cancer cells can be administered to apatient as a cellular vaccine.

[0063] Bystander cells are cells that lack major histocompatibilityclass I (MHC-I) antigens and major histocompatibility class II (MHC-II)antigens on their surface and hence find use as a universal cytokine orcostimulatory molecule-producing cell line. A heterologous nucleic acidcoding sequence or gene for a cytokine or costimulatory molecule may beintroduced into such bystander cells, followed by expression thereof,resulting in production of a recombinant live bystander cell line. Thebystander cell line can be frozen with the cryoprotective medium andmethods described herein. When thawed, the recombinant bystander cellscan be administered to a patient as a cellular vaccine. The use ofbystander cells in active immunotherapy is described in U.S. Pat. No.6,464,973, expressly incorporated by reference herein.

[0064] The recombinant live cancer cells can be cultured in conventionalnutrient media modified as appropriate to the particular cell type.Culture methods and modifications thereof are well within the knowledgeof those of skill in the art. References for cell isolation and cultureinclude Freshney (2000) Culture of Animal Cells, a Manual of BasicTechnique, fourth edition, Wiley- Liss, New York and the referencescited therein; Payne et al. (1993) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg N. Y.)and Atlas and Parks (eds.) The Handbook of Microbiological Media, secondedition (1997) CRC Press, Boca Raton, Fla.

[0065] Kits

[0066] Kits for preserving the viability of recombinant live cancercells are also included in the invention. In one aspect, a kit of theinvention includes: a container containing a cryoprotective mediumcomprising about 5% by weight to about 22% by weight hydroxyethyl starch(HES) or derivative thereof alone or in combination with about 1% byweight to about 15% by weight DMSO, and, optionally, from about 0 toabout 10% human serum albumin (HSA), and instructional materialsteaching the use of the cryoprotective medium to preserve cellviability. In another aspect, a kit of the invention includes: acontainer containing a cryoprotective medium comprising about 5% byweight to about 15% by weight hydroxyethyl starch (HES) or derivativethereof alone or in combination with about 1% by weight to about 10% byweight DMSO, and, optionally, from about 0 to about 10% human serumalbumin (HSA), and instructional materials teaching the use of thecryoprotective medium to preserve cell viability. In yet another aspect,a kit of the invention includes: a container containing a cryoprotectivemedium comprising about 5% by weight to about 10% by weight hydroxyethylstarch (HES) or derivative thereof alone or in combination with about 1%by weight to about 6% by weight DMSO, and, optionally, from about 0 toabout 10% human serum albumin (HSA), and instructional materialsteaching the use of the cryoprotective medium to preserve cellviability.

[0067] A kit can also include a container containing a cryoprotectivemedium comprising about 5% by weight to about 22% by weight hydroxyethylstarch (HES) or derivative thereof, about 3% by weight to about 15% byweight glycerol, and, optionally, from about 0 to about 10% human serumalbumin (HSA), and, instructional materials teaching the use of thecryoprotective medium to preserve cell viability. In another embodiment,a kit can also include a container containing a cryoprotective mediumcomprising about 5% by weight to about 15% by weight hydroxyethyl starch(HES) or derivative thereof, about 3% by weight to about 10% by weightglycerol, and, optionally, from about 0 to about 10% human serum albumin(HSA), and, instructional materials teaching the use of thecryoprotective medium to preserve cell viability. In yet anotherembodiment, a kit can also include a container containing acryoprotective medium comprising about 5% by weight to about 10% byweight hydroxyethyl starch (HES) or derivative thereof, about 3% byweight to about 6% by weight glycerol, and, optionally, from about 0 toabout 10% human serum albumin (HSA), and, instructional materialsteaching the use of the cryoprotective medium to preserve cellviability.

[0068] The HES in the kit can include a variety of molecules asdescribed herein. In one embodiment, HES includes HES, which is, e.g.,(a) etherified with hydroxyethyl groups wherein the degree of molecularsubstitution is about 0.70; (b) etherified with hydroxyethyl groups,wherein the degree of molecular substitution is from about 0.4 to about0.6; or a combination of (a) and (b). In another embodiment, HESincludes a molecule with, e.g., (a) a molecular weight of from about 420to about 420 kDa; (b) a molecular weight of from about 200 to about 290kDa; or a combination of (a) and (b).

[0069] The instructional material can be affixed to the packagingmaterial or can be included as a package insert. While the instructionalmaterial typically comprise written or printed materials they are notlimited to such. Any medium capable of storing such instructionalmaterial and communicating them to an end user is contemplated by thisinvention. Such media include, but are not limited to, electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. As used herein, the term“instructional material” can include the address of an internet sitethat provides the instructions.

[0070] The following examples are offered to illustrate, but not tolimit the claimed invention.

EXAMPLE 1 Viability and Cytokine Production Following Freezing andThawing of Recombinant Live Cancer Cells

[0071] The present invention provides formulations and methods for thecryopreservation of recombinant live cancer cells. The cells may beautologous, allogeneic or bystander cells. The formulations and methodsdescribed herein are applicable to all three types of recombinant livecancer cells. In developing the methods and formulations describedherein, the allogeneic human prostate tumor cell line, LNCaP, wascultured under standard conditosn, pelleted and resuspended in a numberof different cryoprotective media. LNCaP cells were suspended in eachcryoprotective medium, frozen to −80° C., then thawed in a 37° C. waterbath, washed with culture medium. Viability was evaluatedmicroscopically by hemacytometer using the trypan-exclusion method. FIG.1 is a graph showing the % viability of LNCaP cells before (pre-freeze),immediately after (initial thaw) and 24 hours after the cells weresuspended in cryoprotective media, frozen to −80° C. and thawed at 37°C. Viability of greater than about 60% was maintained when thecryoprotective media included HES (either Hetastarch or Pentastarch)together with 5% glycerol and 5% HSA in PBS.

[0072] The viability of LNCaP cells following suspension in polymer 1B(20% HES) with and without 5% glycerol and with or without a secondpolymer was evaluated microscopically by hemacytometer using thetrypan-exclusion method. The suspensions were frozen to −80° C., thawedin a 37° C. water bath, washed with culture medium, then viability wasevaluated immediately upon thawing at 37° C. and 24 h later. The LNCaPcells were suspended in cryoprotective media comprising polymer 1B (20%HES) alone with or without a second polymer (FIG. 2A) or polymer 1B (20%HES) plus 5% glycerol and with or without a second polymer (FIG. 2B).Viability of greater than about 60% was maintained upon initial thaw and24 hours later in each case where20% HES was used in combination with 5%glycerol in the cryoprotective media. The viability and GM-CSF secretionof several independently produced cultures of PC-3 prostateadenocarcinoma cells was evaluated following suspension in 20% HES +5%DMSO or glycerol, and freezing at −80° C. The frozen cell suspensionswere thawed in a 37° C. water bath, washed with culture medium, thenviability was evaluated. One million of the medium-suspended cells wereseeded into a culture flask and allowed to attach over 24 hr. At 24 hr,the medium was replaced. At 48 hr, the medium was collected and measuredfor GM-CSF concentration by ELISA. The amount of secreted GM-CSF wasnormalized to a secretion rate per million cells. The % viability of thePC-3 cells is shown in FIG. 3A and GM-CSF secretion by the cells isshown in FIG. 3B. Greater than 80% viability was maintained when eitherDMSO or glycerol was used in the cryoprotective media and a GM-CSFsecretion level of greater than 150 ng/10⁶ cells/day was observed ineach case.

[0073] The viability and GM-CSF secretion of several independentlyproduced cultures of LNCaP cells was evaluated following suspension in20% HES +5% DMSO or glycerol, and freezing at −80° C. The frozen cellsuspensions were thawed in a 37° C. water bath, washed with culturemedium, then viability was evaluated. One million of themedium-suspended cells were seeded into a culture flask and allowed toattach over 24 hr. At 24 hr, the medium was replaced. At 48 hr, themedium was collected and measured for GM-CSF concentration by ELISA. Theamount of secreted GM-CSF was normalized to a secretion rate per millioncells. The % viability of the LNCaP cells is shown in FIG. 4A and GM-CSFsecretion by the cells is shown in FIG. 4B. Greater than 60% viabilitywas maintained and a GM-CSF secretion level of greater than 10 ng/10⁶cells/day was detected when DMSO was used in the cryoprotective media.

[0074] In a further study, LNCaP cells were suspended in individualcryoprotective media containing 5% glycerol in PBS and from 8% to 26%HES, respectively, then frozen at −80° C. The frozen cell suspensionswere thawed in a 37° C. water bath, washed with culture medium, thenviability was evaluated. The cells were then seeded into a flask withcell culture medium. After 24 hr, the cells were collected and measuredfor viability. FIG. 5 is a graph showing the % viable cell recovery ofthe LNCaP cells immediately following thawing at 37° C. (initial thaw)and 24 h later. The results show that greater than 60% viability wasmaintained when 5% glycerol and from about 10% to 26% HES (Pentastarch)was included in the cryoprotective media.

EXAMPLE 2 Viability and Cytokine Production Following Long Term Freezingand Thawing of Recombinant Live Cancer Cells

[0075] The effect of various cryoprotective media on viability andGM-CSF production by recombinant PC-3 cells was evaluated at time-zero(the time of freezing) and after 3 months, 6 months and 12 monthsstorage at −80° C., respectively. The formulations tested included 24%human serum albumin (HSA), 5% glycerol; 8% HSA, 10% DMSO, 1%Pentastarch; 8% HSA, 10% DMSO; 8% HSA, 10% DMSO, 5% Pentastarch; 8% HSA,5% DMSO; 20% Pentastarch; 10% DMSO; and 20% Pentastarch, 5% DMSO. Atintervals over the 1 year of storage, the frozen cell suspensions werethawed in a 37° C. water bath, washed with culture medium, then measuredfor viability. One million of the medium-suspended cells were seededinto a culture flask and allowed to attach over 24 hr. At 24 hr, themedium was replaced. At 48 hr, the medium was collected and measured forGM-CSF concentration by ELISA. The amount of secreted GM-CSF wasnormalized to a secretion rate per million cells. The % viability of thecells is shown in FIG. 6A and GM-CSF secretion by the cells is shown inFIG. 6B. Greater than 60% viability was maintained with eachcryoprotective media tested. The most consistent GM-CSF secretion overtime was detected when 5 or 10% DMSO was used in combination withPentastarch with or without HSA in the cryoprotective media (greaterthan 200 ng GM-CSF/10⁶ cells/day).

[0076] All publications, patents, and patent applications cited hereinare hereby incorporated by reference in their entirety for all purposes.While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments can be used and that it isintended that the invention be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the invention as defined bythe following claims.

What is claimed is:
 1. A composition comprising a recombinant livecancer cell in a cryoprotective medium, wherein the cryoprotectivemedium comprises about 5% by weight to about 22% by weight of ahydroxyethyl starch (HES) or derivative thereof and about 1% by weightto about 15% by weight of DMSO.
 2. The composition of claim 1, furthercomprising from about 0% by weight to about 10% by weight human serumalbumin (HSA).
 3. The composition of claim 2, wherein the HES comprisesHES: (a) etherified with hydroxyethyl groups wherein the degree ofmolecular substitution is from about 0.60-0.80; (b) etherified withhydroxyethyl groups, wherein the degree of molecular substitution isfrom about 0.40-0.60; or a combination of (a) and (b).
 4. Thecomposition of claim 3, wherein the HES comprises HES: (a) etherifiedwith hydroxyethyl groups wherein the degree of molecular substitution isabout 0.70; (b) etherified with hydroxyethyl groups, wherein the degreeof molecular substitution is from 0.4 to about 0.5; or a combination of(a) and (b).
 5. The composition of claim 2, wherein the HES comprisesHES with: (a) a molecular weight of from about 420 to about 420 kDa; (b)a molecular weight of from about 200 to about 290 kDa; or a combinationof (a) and (b).
 6. The composition of claim 2, wherein the recombinantlive cancer cell comprises an introduced heterologous nucleic acidcoding sequence for a cytokine or costimulatory molecule.
 7. Thecomposition of claim 6, wherein the cytokine is granulocyte-macrophagecolony stimulating factor (GM-CSF).
 8. The composition of claim 2,wherein the cancer cell is selected from the group consisting of a lungcancer cell, a pancreatic cancer cell, a prostate cancer cell, a kidneycancer cell, a myeloma cell, and a leukemic cell.
 9. The composition ofclaim 2, wherein the recombinant live cancer cell composition comprisespatient-derived tumor cells.
 10. The composition of claim 9, whereinsaid recombinant live cancer cell is autologous.
 11. The composition ofclaim 2, wherein said recombinant live cancer cell is allogeneic. 12.The composition of claim 2, wherein said recombinant live cancer cell isa bystander cell.
 13. A composition comprising a recombinant live cancercell in a cryoprotective medium, wherein the cryoprotective mediumcomprises about 8% by weight HES, about 2% by weight HSA, and about 5%by weight DMSO.
 14. A composition comprising a recombinant live cancercell in a cryoprotective medium, wherein the cryoprotective mediumcomprises about 5% by weight to about 22% by weight of the ahydroxyethyl starch (HES) or derivative thereof and about 1% by weightto about 15% by weight of the glycerol, said composition furthercomprising from about 0 to about 10% human serum albumin (HSA).
 15. Thecomposition of claim 14, wherein the HES comprises HES: (a) etherifiedwith hydroxyethyl groups wherein the degree of molecular substitution isfrom about 0.60-0.80; (b) etherified with hydroxyethyl groups, whereinthe degree of molecular substitution is from about 0.40-0.60; or acombination of (a) and (b).
 16. The composition of claim 15, wherein theHES comprises HES: (a) etherified with hydroxyethyl groups wherein thedegree of molecular substitution is about 0.70; (b) etherified withhydroxyethyl groups, wherein the degree of molecular substitution isfrom 0.4 to about 0.5; or a combination of (a) and (b).
 17. Thecomposition of claim 14, wherein the HES comprises HES with: (a) amolecular weight of from about 420 to about 420 kDa; (b) a molecularweight of from about 200 to about 290 kDa; or a combination of (a) and(b).
 18. The composition of claim 14, wherein the recombinant livecancer cell comprises an introduced heterologous nucleic acid codingsequence for a cytokine or costimulatory molecule.
 19. The compositionof claim 18, wherein the cytokine is granulocyte-macrophage colonystimulating factor (GM-CSF).
 20. The composition of claim 14, whereinthe cancer cell is selected from the group consisting of a lung cancercell, a pancreatic cancer cell, a prostate cancer cell, a kidney cancercell, a myeloma cell, and a leukemic cell.
 21. The composition of claim14, wherein said recombinant live cancer cell composition comprisespatient-derived tumor cells.
 22. The composition of claim 21, whereinsaid recombinant live cancer cell is autologous.
 23. The composition ofclaim 14, wherein said recombinant live cancer cell is allogeneic. 24.The composition of claim 14, wherein said recombinant live cancer cellis a bystander cell.
 25. A composition comprising a recombinant livecancer cell in a cryoprotective medium, wherein the cryoprotectivemedium comprises about 8% by weight HES, about 2% by weight HSA, andabout 5% by weight glycerol.
 26. A method for preserving viability ofrecombinant live cancer cells, the method comprising: obtaining aplurality of recombinant live cancer cells; concentrating the pluralityof recombinant live cancer cells; suspending said recombinant livecancer cells in a cryoprotective medium, wherein the cryoprotectivemedium comprises 5% by weight to about 22% by weight of a hydroxyethylstarch (HES) or derivative thereof and about 1% by weight to about 15%by weight of DMSO, thereby providing a suspension; and, freezing thesuspension to a cryogenic frozen state, thereby providing a frozensuspension, wherein following thawing of the suspension to a liquidstate at least about 60% of the recombinant live cancer cells remainviable.
 27. The method of claim 26, further comprising irradiating therecombinant live cancer cells.
 28. The method of claim 27, wherein therecombinant live cancer cells are irradiated prior to freezing.
 29. Themethod of claim 27, wherein the recombinant live cancer cells areirradiated in the cryogenic frozen state.
 30. The method of claim 26,wherein the frozen suspension is frozen at—a temperature of from about−200° C. to a temperature of about −35° C.
 31. The method of claim 26,further comprising: thawing the frozen suspension thereby providing athawed suspension and administering the thawed suspension to a subject,wherein the thawed suspension is not washed prior to the administeringstep.
 32. The method of claim 26, wherein the cryoprotective mediumfurther comprises about 0% by weight to about 10% by weight human serumalbumin (HSA).
 33. The method of claim 32, wherein the recombinant livecancer cells are mammalian cells selected from the group consisting oflung cancer cells, pancreatic cancer cells, prostate cancer cells,kidney cancer cells, myeloma cells, and leukemic cells.
 34. A frozensuspension prepared by the method of claim
 33. 35. A thawed suspensionprepared by the method of claim
 33. 36. The method of claim 33, whereinthe plurality of recombinant live cancer cells are autologous orallogeneic and further comprise an introduced heterologous nucleic acidcoding sequence for granulocyte-macrophage colony stimulating factor(GM-CSF).
 37. The method of claim 33, wherein the recombinant livecancer cells are bystander cells, said method further comprising thawingthe frozen suspension, thereby providing a thawed suspension; providingpatient-derived tumor cells, mixing the thawed suspension with thepatient-derived tumor cells, thereby providing a mixed suspension; and,administering the mixed suspension to a subject.
 38. The method of claim37, wherein the patient-derived tumor cells comprise irradiated tumorcells.
 39. The method of claim 32, wherein the cryoprotective mediumcomprises about 8% by weight HES, about 2% by weight HSA, and about 5%by weight DMSO.
 40. The method of claim 39, further comprising: thawingthe frozen suspension, thereby providing a thawed suspension; and,administering the thawed suspension to a subject wherein the thawedsuspension is not washed prior to the administering step.
 41. The methodof claim 40, wherein said recombinant live cancer cell is autologous.42. The method of claim 40, wherein said recombinant live cancer cell isallogeneic.
 43. The method of claim 40, wherein said recombinant livecancer cell is a bystander.
 44. A thawed suspension prepared by themethod of claim
 40. 45. A kit for preserving cell viability comprising:a container containing a cryoprotective medium comprising about 5% byweight to about 22% by weight hydroxyethyl starch (HES) or derivativethereof alone or in combination with about 1% by weight to about 15% byweight DMSO; from about 0 to about 10% human serum albumin (HSA), andinstructional materials teaching the use of the cryoprotective medium topreserve cell viability.
 46. The kit of claim 45, wherein the HEScomprises HES: (a) etherified with hydroxyethyl groups wherein thedegree of molecular substitution is about 0.70; (b) etherified withhydroxyethyl groups, wherein the degree of molecular substitution isfrom 0.4 to about 0.5; or a combination of (a) and (b).
 47. The kit ofclaim 45, wherein the HES comprises HES with: (a) a molecular weight offrom about 420 to about 420 kDa; (b) a molecular weight of from about200 to about 290 kDa; or a combination of (a) and (b).
 48. A kit forpreserving cell viability comprising: a container containing acryoprotective medium comprising about 5% by weight to about 22% byweight hydroxyethyl starch (HES) or derivative thereof, about 3% byweight to about 15% by weight glycerol; from about 0 to about 10% humanserum albumin (HSA), and, instructional materials teaching the use ofthe cryoprotective medium to preserve cell viability.
 49. The kit ofclaim 48, wherein the HES comprises HES: (a) etherified withhydroxyethyl groups wherein the degree of molecular substitution isabout 0.70; (b) etherified with hydroxyethyl groups, wherein the degreeof molecular substitution is from 0.4 to about 0.5; or a combination of(a) and (b).
 50. The kit of claim 48, wherein the HES comprises HESwith: (a) a molecular weight of from about 420 to about 420 kDa; (b) amolecular weight of from about 200 to about 290 kDa; or a combination of(a) and (b).